Method for non-invasive blood analyte measurement with improved optical interface

ABSTRACT

A method for non-invasively measuring the concentration of an analyte, particularly blood analyte in blood. The method utilizes spectrographic techniques in conjunction with an improved optical interface between a sensor probe and a skin surface or tissue surface of the body containing the blood to be analyzed. An index-matching medium is disclosed to improve the interface between the sensor probe and skin surface during spectrographic analysis. In a preferred embodiment, the blood analyte concentration in blood is quantified utilizing a partial squares analysis relative to a model incorporating analysis of plural known blood samples.

CROSS REFERENCES TO CO-PENDING APPLICATIONS

This Application is a continuation of application Ser. No. 09/174,812,filed Oct. 19, 1998, now U.S. Pat. No. 6,152,876, which is aContinuation-in-part of U.S. Patent Application Ser. No. 08/844,501,filed Apr. 18, 1997, entitled “Method for Non-Invasive Blood AnalyteMeasurement with Improved Optical Interface”, now U.S. Pat. No.5,823,951, issued Oct. 20, 1998, to the same assignee as the presentapplication which is a continuation of Ser. No. 08/512,940 filed Aug. 9,1995 now U.S. Pat. No. 5,655,530.

TECHNICAL FIELD

The present invention relates generally to a non-invasive method formeasuring a blood analyte, particularly glucose, utilizing spectroscopicmethods. More particularly, the method incorporates an improved inputoptical interface for irradiating biological tissue with infrared energyhaving at least several wavelengths and an improved output opticalinterface for receiving non-absorbed infrared energy as a measure ofdifferential absorption by the biological sample to determine an analyteconcentration. An index-matching medium is disclosed as a key element ofthe improved optical interface.

BACKGROUND OF THE INVENTION

The need and demand for an accurate, non-invasive method for determiningblood glucose level in patients is well documented. Barnes et al. (U.S.Pat. No. 5,379,764) disclose the necessity for diabetics to frequentlymonitor glucose levels in their blood. It is further recognized that themore frequent the analysis, the less likely there will be large swingsin glucose levels. These large swings are associated with the symptomsand complications of the disease, whose long term effects can includeheart disease, arteriosclerosis, blindness, stroke, hypertension, kidneyfailure, and premature death. As described below, several systems havebeen proposed for the non-invasive measurement of glucose in blood.However, despite these efforts a lancet cut into the finger is stillnecessary for all presently commercially available forms of home glucosemonitoring. This is believed so compromising to the diabetic patientthat the most effective use of any form of diabetic management is rarelyachieved.

The various proposed non-invasive methods for determining blood glucoselevel, discussed individually below, generally utilize quantitativeinfrared spectroscopy as a theoretical basis for analysis. Infraredspectroscopy measures the electromagnetic radiation (0.7-25 μm) asubstance absorbs at various wavelengths. Molecules do not maintainfixed positions with respect to each other, but vibrate back and forthabout an average distance. Absorption of light at the appropriate energycauses the molecules to become excited to a higher vibration level. Theexcitation of the molecules to an excited state occurs only at certaindiscrete energy levels, which are characteristic for that particularmolecule. The most primary vibrational states occur in the mid-infraredfrequency region (i.e., 2.5-25 μm). However, non-invasive analytedetermination in blood in this region is problematic, if not impossible,due to the absorption of the light by water. The problem is overcomethrough the use of shorter wavelengths of light which are not asattenuated by water. Overtones of the primary vibrational states existat shorter wavelengths and enable quantitative determinations at thesewavelengths.

It is known that glucose absorbs at multiple frequencies in both themid- and near-infrared range. There are, however, other infrared activeanalytes in the blood which also absorb at similar frequencies. Due tothe overlapping nature of these absorption bands, no single or specificfrequency can be used for reliable non-invasive glucose measurement.Analysis of spectral data for glucose measurement thus requiresevaluation of many spectral intensities over a wide spectral range toachieve the sensitivity, precision, accuracy, and reliability necessaryfor quantitative determination. In addition to overlapping absorptionbands, measurement of glucose is further complicated by the fact thatglucose is a minor component by weight in blood, and that the resultingspectral data may exhibit a non-linear response due to both theproperties of the substance being examined and/or inherentnon-linearities in optical instrumentation.

A further common element to non-invasive glucose measuring techniques isthe necessity for an optical interface between the body portion at thepoint of measurement and the sensor element of the analyticalinstrument. Generally, the sensor element must include an input elementor means for irradiating the sample point with infrared energy. Thesensor element must further include an output element or means formeasuring transmitted or reflected energy at various wave lengthsresulting from irradiation through the input element.

Robinson et al. (U.S. Pat. No. 4,975,581) disclose a method andapparatus for measuring a characteristic of unknown value in abiological sample using infrared spectroscopy in conjunction with amultivariate model that is empirically derived from a set of spectra ofbiological samples of known characteristic values. The above-mentionedcharacteristic is generally the concentration of an analyte, such asglucose, but also may be any chemical or physical property of thesample. The method of Robinson et al. involves a two-step process thatincludes both calibration and prediction steps. In the calibration step,the infrared light is coupled to calibration samples of knowncharacteristic values so that there is differential attenuation of atleast several wavelengths of the infrared radiation as a function of thevarious components and analytes comprising the sample with knowncharacteristic value. The infrared light is coupled to the sample bypassing the light through the sample or by reflecting the light from thesample. Absorption of the infrared light by the sample causes intensityvariations of the light that are a function of the wavelength of thelight. The resulting intensity variations at the at least severalwavelengths are measured for the set of calibration samples of knowncharacteristic values. Original or transformed intensity variations arethen empirically related to the known characteristic of the calibrationsamples using a multivariate algorithm to obtain a multivariatecalibration model. In the prediction step, the infrared light is coupledto a sample of unknown characteristic value, and the calibration modelis applied to the original or transformed intensity variations of theappropriate wavelengths of light measured from this unknown sample. Theresult of the prediction step is the estimated value of thecharacteristic of the unknown sample. The disclosure of Robinson et al.is incorporated herein by reference.

Several of the embodiments disclosed by Robinson et al. are non-invasiveand incorporate an optical interface having a sensor element. Asdepicted in FIGS. 5 and 6 of Robinson et al., the optical interfaceincludes first, an input element and second, an output element. Theinput element is an infrared light source or near infrared light source.The input element interface with the sample or body portion containingblood to be tested includes transmitting the light energy or propagatingthe light energy to the surface of the skin via the air. The outputelement includes a detector which receives the transmitted or reflectedlight energy. The output interface with the sample also includespropagating the transmitted or reflected light through the air from theskin.

Barnes et al. (U.S. Pat. No. 5,379,764) disclose a spectrographic methodfor analyzing glucose concentration, wherein near infrared radiation isprojected on a portion of the body, the radiation including a pluralityof wavelengths, followed by sensing the resulting radiation emitted fromthe portion of the body as affected by the absorption of the body. Themethod disclosed includes pretreating the resulting data to minimizeinfluences of offset and drift to obtain an expression of the magnitudeof the sensed radiation as modified.

The sensor element disclosed by Barnes et al. includes a dual conductorfiber optic probe which is placed in contact or near contact with theskin of the body. The first conductor of the dual conductor fiber opticprobe acts as an input element which transmits the near infraredradiation to the skin surface while in contact therewith. The secondconductor fiber of the dual conductor probe acts as an output elementwhich transmits the reflected energy or non-absorbed energy back to aspectrum analyzer. The optical interface between the sensor element andthe skin is achieved by simply contacting the skin surface with theprobe, and can include transmitting the light energy through air to theskin and through air back to the probe depending upon the degree ofcontact between the probe and skin. Irregularities in the skin surfaceand at the point of measurement will affect the degree of contact.

Dähine et al. (U.S. Pat. No. 4,655,225) disclose the employment of nearinfrared spectroscopy for non-invasively transmitting optical energy inthe near infrared spectrum through a finger or earlobe of a subject.Also discussed is the use of near infrared energy diffusely reflectedfrom deep within the tissues. Responses are derived at two differentwavelengths to quantify glucose in the subject. One of the wavelengthsis used to determine background absorption, while the other wavelengthis used to determine glucose absorption.

The optical interface disclosed by Dähne et al. includes a sensorelement having an input element which incorporates a directive lightmeans which is transmitted through the air to the skin surface. Thelight energy which is transmitted or reflected from the body tissue as ameasure of absorption is received by an output element. The interfacefor the output element includes transmitting the reflected ortransmitted light energy through air to the detector elements.

Caro (U.S. Pat. No. 5,348,003) discloses the use of temporally-modulatedelectromagnetic energy at multiple wavelengths as the irradiating lightenergy. The derived wavelength dependence of the optical absorption perunit path length is compared with a calibration model to deriveconcentrations of an analyte in the medium.

The optical interface disclosed by Caro includes a sensor element havingan input element, wherein the light energy is transmitted through afocusing means onto the skin surface. The focusing means may be near orin contact with the skin surface. The sensor element also includes anoutput element which includes optical collection means which may be incontact with the skin surface or near the skin surface to receive lightenergy which is transmitted through the tissue. Again, a portion of thelight energy is propagated through air to the skin surface and back tothe output element due to non-contact with the sensor and irregularitiesin the skin surface.

Problems with the optical interface between the tissue and theinstrument have been recognized. In particular, optical interfaceproblems associated with coupling light into and back out of the tissuewere recognized by Ralf Marbach as published in a thesis entitled“MeBverfahren zur IR-spektroskopishen Blutglucose Bestimmung” (Englishtranslation “Measurement Techniques for IR Spectroscopic Blood GlucoseDetermination”), published in 1993.

Marbach states that the requirements of the optical accessory formeasurement of the diffuse reflection of the lip are:

1) High optical “throughput” for the purpose of optimizing the S/N ratioof the spectra,

2) Suppression of the insensitivity to Fresnel or specular reflection onthe skin surface area.

The measurement accessory proposed by Marbach attempts to meet bothrequirements through the use of a hemispherical immersion lens. The lensis made out of a material which closely matches the refractive index oftissue, calcium fluoride. As stated by Marbach, the important advantagesof the immersion lens for transcutaneous diffuse reflection measurementsare the nearly complete matching of the refraction indices of CaF₂ andskin and the successful suppression of the Fresnel reflection.

Calcium fluoride, however is not an ideal index match to tissue, havingan index of 1.42, relative to that of tissue, at approximately 1.38.Thus, an index mismatch occurs at the lens to tissue interface assumingcomplete contact between the lens and tissue. The optical efficiency ofthe sampling accessory is further compromised by the fact that the lensand the tissue will not make perfect optical contact due to roughness ofthe tissue. The result is a significant refractive index mismatch wherethe light is forced to travel from the lens (N=1.42) to air (N=1.0) totissue (N=1.38). Thus, the inherent roughness of tissue results in smallair gaps between the lens and the tissue, which decrease the opticalthroughput of the system, and subsequently compromise the performance ofthe measurement accessory.

The magnitude of the problem associated with refractive index mismatchis a complicated question. First, a fraction of light, which wouldotherwise be available for spectroscopic analysis of blood analytes,gets reflected at the mismatch boundary and returns to the input orcollection optical system without interrogating the sample. The effectis$R = \frac{\left( {N^{\prime} - N} \right)^{2}}{\left( {N^{\prime} + N} \right)^{2}}$

governed by the Fresnel Equation:

For normally incident, randomly polarized light, where N and N′ are therefractive indices of the two media. Solving for the air/CaF, interfacegives an R=0.03, or a 3% reflection. This interface must be traversedtwice, leading to a 6% reflected component which does not interrogatethe sample. These interface mismatches are multiplicative. The fractionof light successfully entering the tissue then must be considered. Insome regions of the spectrum, for instance, under a strong water band,almost all of the transmitted light gets absorbed by the tissue. Theresult is that this seemingly small reflected light component from therefractive index mismatch can virtually swamp out and obscure thedesired signal from the sample.

Finally, it is useful to consider the critical angle effect as lightattempts to exit the tissue. Tissue is highly scattering and so a lightray which launches into tissue at normal incidence may exit the tissueat a high angle of incidence. If the coupling lens is not in intimatecontact with the tissue, these high angle rays will be lost to totalinternal reflection. The equation which defines the critical angle, orthe point of total internal reflection, is as$\Theta_{c} = {\sin^{- 1}\quad \left( \frac{N}{N^{\prime}} \right)}$

follows:

When light is propagating through a higher index material like tissue(N′=1.38) and approaching an interface with lower refractive index likeair (N=1.0), a critical angle of total internal reflection occurs. Lightapproaching such an interface at greater than the critical angle willnot propagate into the rarer medium (air), but will totally internallyreflect back into the tissue. For the aforementioned tissue/airinterface, the critical angle is 46.4. No light steeper than this anglewould escape. Intimate, optical contact is therefore essential toefficient light capture from tissue.

As detailed above, each of the prior art apparatus for non-invasivelymeasuring glucose concentration utilize a sensor element. Each sensorelement includes an input element and an output element. The opticalinterface between the input element, output element and the skin surfaceof the tissue to be analyzed in each apparatus is similar. In eachinstance, the input light energy is transmitted through air to thesurface or potentially through air due to a gap in the contact surfacebetween the input sensor and the skin surface. Likewise, the outputsensor receives transmitted or reflected light energy via transmissionthrough air to the output sensor, or potentially through a gap betweenthe sensor element and the skin surface even though attempts are made toplace the output sensor in contact with the skin. It is believed thatthe optical interfaces disclosed in the prior art affect the accuracyand consistency of the data acquired utilizing the prior art methods andapparatus. Thus, the accuracy of these methods for non-invasivelymeasuring glucose are compromised.

Wu et al. (U.S. Pat. No. 5,452,723) disclose a method of spectrographicanalysis of a tissue sample, which includes measuring the diffusereflectance spectrum, as well as a second selected spectrum, such asfluorescence, and adjusting the spectrum with the reflectance spectrum.Wu et al. assert that this procedure reduces the sample-to-samplevariability. Wu et al. disclose the use of an optical fiber as an inputdevice that is bent at an acute angle so that incident light from thefiber impinges on an optically smooth surface of an optical couplingmedium. The optical coupling medium is indexed matched to the tissue sothat little or no specular reflection occurs at the interface betweenthe catheter and the tissue. Wu et al. further disclose that thecatheter can be used in contact or non-contact modes with the tissue. Incontact mode, the end of the catheter is placed in direct contact withthe tissue to accomplish index matched optical coupling. Thus, theoptical coupling medium of Wu et al. is a solid end portion on theoptical fiber. Wu et al. further disclose that the catheter can be usedin a non-contact mode, wherein the gap left between the end of thecatheter and the tissue can be filled with an index-matched fluid toprevent specular reflections. The only criteria disclosed throughout theWu et al. specification for the fluid is that it is index matched toprevent specular reflections, which is only one aspect of an optimumoptical interface for spectrographic analysis of an analyte in blood.

Accordingly, the need exists for a method and apparatus fornon-invasively measuring glucose concentrations in blood whichincorporates an improved optical interface. The optical interface shouldproduce consistent repeatable results so that the analyte concentrationcan be accurately calculated from a model such as that disclosed byRobinson et al. The optical interface should maximize both the input andoutput light energy from the source into the tissue and from the tissueback to the output sensor. The detrimental effects of gaps due toirregularities in the surface of the skin or the presence of othercontaminants should be reduced or eliminated. Means should also beprovided to guarantee that such optimized interface is achieved eachtime a user is coupled to the device for analysis.

The present invention addresses these needs as well as other problemsassociated with existing methods for non-invasively measuring glucoseconcentration in blood utilizing infrared spectroscopy and the opticalinterface associated therewith. The present invention also offersfurther advantages over the prior art and solves problems associatedtherewith.

SUMMARY OF THE INVENTION

The present invention is a method for non-invasively measuring theconcentration of an analyte, particularly glucose in human tissue. Themethod utilizes spectroscopic techniques in conjunction with an improvedoptical interface between a sensor probe and a skin surface or tissuesurface of the body containing the tissue to be analyzed.

The method for non-invasively measuring the concentration of glucose inblood includes first providing an apparatus for measuring infraredabsorption by an analyte containing tissue. The apparatus includesgenerally three elements, an energy source, a sensor element, and aspectrum analyzer. The sensor element includes an input element and anoutput element. The input element is operatively connected to the energysource by a first means for transmitting infrared energy. The outputelement is operatively connected to the spectrum analyzer by a secondmeans for transmitting infrared energy.

In preferred embodiments, the input element and output element compriselens systems which focus the infrared light energy to and from thesample. In a preferred embodiment, the input element and output elementcomprise a single lens system which is utilized for both input ofinfrared light energy from the energy source and output of both specularand diffusely reflected light energy from the analyte-containing sample.

Alternatively, the input element and output element can comprise twolens systems, placed on opposing sides of an analyte-containing sample,wherein light energy from the energy source is transmitted to the inputelement and light energy transmitted through the analyte-containingsample then passes through the output element to the spectrum analyzer.

The first means for transmitting infrared energy, in preferredembodiments, simply includes placing the infrared energy sourceproximate to the input element so that light energy from the source istransmitted via the air to the input element. Further, in preferredembodiments, the second means for transmitting infrared energypreferably includes a single mirror or system of mirrors which directthe light energy exiting the output element through the air to thespectrum analyzer.

In practicing the method of the present invention, an analyte containingtissue area is selected as the point of analysis. This area can includethe skin surface on the finger, earlobe, forearm or any other skinsurface. Preferably, the analyte-containing tissue in the area forsampling includes blood vessels near the surface and a relativelysmooth, uncalloused skin surface. A preferred sample location is theunderside of the forearm.

A quantity of an index-matching medium or fluid is then placed on theskin area to be analyzed. The index-matching fluid detailed herein isselected to optimize introduction of light into the tissue, reducespecular light and effectively get light out of the tissue. The mediumor fluid preferably contains an additive which confirm proper couplingto the skin surface by a proper fluid, thus assuring the integrity oftest data. It is preferred that the index-matching medium is non-toxicand has a spectral signature in the near infrared region which isminimal, and is thus minimally absorbing of light energy havingwavelengths relevant to the analyte being measured. In preferredembodiments, the index-matching medium has a refractive index of about1.38. Further, the refractive index of the medium should be constantthroughout the composition. The composition of the index-matching mediumis detailed below.

The sensor element, which includes the input element and the outputelement, is then placed in contact with the index-matching medium.Alternatively, the index-matching medium can be first placed on thesensor element, followed by placing the sensor element in contact withthe skin with the index-matching medium disposed therebetween. In thisway, the input element and output element are coupled to the analytecontaining tissue or skin surface via the index-matching medium whicheliminates the need for the light energy to propagate through air orpockets of air due to irregularities in the skin surface.

In analyzing for the concentration of glucose in the analyte containingtissue, light energy from the energy source is transmitted via the firstmeans for transmitting infrared energy into the input element. The lightenergy is transmitted from the input element through the index-matchingmedium to the skin surface. Some of the light energy contacting theanalyte-containing sample is differentially absorbed by the variouscomponents and analytes contained therein at various depths within thesample. Some of the light energy is also transmitted through the sample.However, a quantity of light energy is reflected back to the outputelement. In a preferred embodiment, the non-absorbed or non-transmittedlight energy is reflected back to the output element upon propagatingthrough the index-matching medium. This reflected light energy includesboth diffusely reflected light energy and specularly reflected lightenergy. Specularly reflected light energy is that which reflects fromthe surface of the sample and contains little or no analyte information,while diffusely reflected light energy is that which reflects fromdeeper within the sample, wherein the analytes are present.

In preferred embodiments, the specularly reflected light energy isseparated from the diffusely reflected light energy. The non-absorbeddiffusely reflected light energy is then transmitted via the secondmeans for transmitting infrared energy to the spectrum analyzer. Asdetailed below, the spectrum analyzer preferably utilizes a computer togenerate a prediction result utilizing the measured intensities, acalibration model, and a multivariate algorithm.

A preferred device for separating the specularly reflected light fromthe diffusely reflected light is a specular control device as disclosedin co-pending and commonly assigned application Ser. No. 08/513,094,filed on Aug. 9, 1995, and entitled “Improved Diffuse ReflectanceMonitoring Apparatus”, now U.S. Pat. No. 5,636,633, issued Jun. 10,1997. The above patent disclosure is hereby incorporated by reference.

In an alternative embodiment, the input element is placed in contactwith a first quantity of index-matching medium on a first skin surface,while the output element is placed in contact with a second quantity ofindex-matching medium on an opposing skin surface. Alternatively, theindex-matching medium can be placed on the input and output elementsprior to skin contact so that the medium is disposed between theelements and the skin surface during measurement. With this alternativeembodiment, the light energy propagated through the input element andfirst quantity of index-matching medium is differentially absorbed bythe analyte containing tissue or reflected therefrom, while a quantityof the light energy at various wavelengths is transmitted through theanalyte containing tissue to the opposing or second skin surface. Fromthe second skin surface, the non-absorbed light energy is propagatedthrough the second quantity of index-matching medium to the outputelement with subsequent propagation to the spectrum analyzer forcalculation of the analyte concentration.

The index-matching medium of the present invention is a key to theimproved accuracy and repeatability of the method described above. Theindex-matching medium is preferably a composition containingchlorofluorocarbons. The composition can also contain perfluorocarbons.One preferred index-matching medium is a fluoronated-chloronatedhydrocarbon polymer oil manufactured by Oxidant Chemical under thetradename FLUOROLUBE.

It has been found that the index-matching mediums of the presentinvention optimize the analysis of a blood analyte in human tissue byeffectively introducing light into the tissue, reducing specular light,and effectively getting light back out of the tissue, which has beendiffusely reflected from analyte-containing areas of the tissue, back tothe output device. This requires selection of an index-matching mediumthat not only has the proper refractive index, but also has minimalabsorption of infrared energy at wavelengths which are relevant to themeasurement of the analyte of interest. Therefore, a preferredindex-matching medium of the present invention is minimally oressentially non absorbing of light energy in the near infrared range ofthe spectrum.

In preferred embodiments, the index-matching medium of the presentinvention also includes a diagnostic additive. The diagnostic additivein the index-matching fluid allows a determination of the height of thefluid layer and/or provides a wavelength calibration for the instrument.These additives allow for assessment of the quality of the lens/tissueinterface and assessment of instrument performance each time anindividual is tested utilizing the apparatus of the present invention.The diagnostic additive can account for about 0.2% to about 20% byweight of the overall fluid. In an alternative embodiment, theindex-matching medium and the diagnostic additive can comprise the samecompound which serves both functions.

The index-matching medium of the present invention can also includephysiological additives which enhance or alter the physiology of thetissue to be analyzed. In particular, preferred physiological additivesinclude vasodilating agents which decrease the equilibration timebetween capillary blood glucose concentration and skin interstitialfluid glucose concentrations to provide a more accurate blood glucosenumber. The physiological additives can account for about 0.2% to about20% by weight of the overall fluid.

The compound can also contain other additives such as a hydrophilicadditive like isopropyl alcohol. The hydrophilic compound is believed totie up the moisture in the skin surface to improve the interface betweenthe fluid and skin. Further, the index-matching medium can containcleansing agents to bind the oil in the skin at the sample point andreduce the effect thereof. Finally, a surfactant can also be included inthe fluid composition. The surfactant improves the wetting of thetissue, creating a uniform interface. An antiseptic material can also beadded to the index-matching medium.

In an alternative embodiment of the current invention, the indexmatching between the optical sensor elements and the tissue can beperformed by a deformable solid. The deformable solid can alter itsshape such that air gaps, due in part to the uneven surfaces of theskin, are minimized. Deformable solids can include at least gelatin,adhesive tape, and substances that are liquid upon application butbecome solid over time.

The index-matching medium preferably has a refractive index of between1.30-1.45, more preferably between 1.35-1.40. Utilization of arefractive index in this range has been found to improve therepeatability and accuracy of the above method by improving opticalthroughput and decreasing spectroscopic variations unrelated to analyteconcentration. Further, the index-matching medium should have aconsistent refractive index throughout the composition. For example, noair bubbles should be present which cause changes in light direction.

In a preferred embodiment, the concentration of glucose in the tissue isdetermined by first measuring the light intensity received by the outputsensor. These measured intensities in combination with a calibrationmodel are utilized by a multivariate algorithm to predict the glucoseconcentration in the tissue. The calibration model empirically relatesthe known glucose concentrations in a set of calibration samples to themeasured intensity variations obtained from said calibration samples. Ina preferred embodiment, the multivariate algorithm used is the partialleast squares method, although other multivariate techniques can beemployed.

The use of an index-matching medium to couple the optical sensor's inputelement and output element to the skin surface reduces the likelihoodthat aberrant data will be acquired. The index-matching medium increasesthe repeatability and accuracy of the measuring procedure. Adverseeffects on the input and output light energy by transmission through airor uneven surfaces of the skin having pockets of air are eliminated.

These and various other advantages and features of novelty whichcharacterize the present invention are pointed out with particularity inthe claims annexed hereto and forming a part hereof. However, for abetter understanding of the invention, its advantages, and the objectobtained by its use, reference should be made to the drawings which forma further part hereof, and to the accompanying descriptive matter inwhich there are illustrated and described preferred embodiments of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, in which like reference numerals indicate correspondingparts or elements of preferred embodiments of the present inventionthroughout the several views.

FIG. 1 is a partial cross-sectional view of a sensor element coupled tothe skin surface via an indexing-matching fluid;

FIG. 2 is a partial cross-sectional view of an alternative embodiment ofa sensor element coupled to opposite sides of a skin surface via anindexing-matching fluid; and

FIG. 3 is a graphical representation of experimental data showing theimprovement in accuracy and repeatability of a sensor coupled to theskin via an index-matching medium.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Detailed embodiments of the present invention are disclosed herein.However, it is to be understood that the disclosed embodiments aremerely exemplary of the present invention which may be embodied invarious systems. Therefore, specific details disclosed herein are not tobe interpreted as limiting, but rather as a basis for the claims and asa representative basis for teaching one of skill in the art to variouslypractice the invention.

The present invention is directed to a method for non-invasivemeasurement of tissue constituents using spectroscopy. It has been foundthat the sample is a complex matrix of materials with differingrefractive indices and absorption properties. Further, because the bloodconstituents of interest are present at very low concentrations, it hasbeen found to be imperative to couple light into and out from the tissuein an efficient manner. The method of the present invention incorporatesan index-matching medium, fluid or deformable solid, to improve theefficiency of coupling the light both into and out of the tissue sample.

The present invention utilizes light energy in the near-infrared regionof the optical spectrum as an energy source for analysis. Water is byfar the largest contributor to absorption in tissue in the near-infraredregion because of its concentration, as well as its strong absorptioncoefficient. It has been found that the total absorption spectrum oftissue, therefore, closely resembles the water spectrum. Less than 0.1percent of the absorption of light is from, for instance, a constituentsuch as glucose. It has been further found that tissue greatly scatterslight because there are many refractive index discontinuities in atypical tissue sample. Water is perfused through the tissue, with arefractive index of 1.33. Cell walls and other features of tissue haverefractive indices closer to 1.5 to 1.6. These refractive indexdiscontinuities give rise to scatter. Although these refractive indexdiscontinuities are frequent, they are also typically small in magnitudeand the scatter generally has a strong directionality towards theforward direction.

This forward scatter has been described in terms of anisotropy, which isdefined as the cosine of the average scatter angle. Thus, for completebackwards scatter, meaning that all scatter events would cause a photonto divert its direction of travel by 180 degrees, the anisotropy factoris −1. Likewise, for complete forward scatter, the anisotropy factor is+1. In the near infrared, tissue has been found to have an anisotropyfactor of around 0.9 to 0.95, which is very forward scattering. Forinstance, an anisotropy factor of 0.9 means that an average photon oflight only scatters through an angle of up to 25 degrees as it passesthrough the sample.

In analyzing for an analyte in tissue, measurements can be made in atleast two different modes. It is recognized that one can measure lighttransmitted through a section of tissue, or one may measure lightreflected or remitted from tissue. It has been recognized thattransmission is the preferred method of analysis in spectroscopy becauseof the forward scattering of light as it passes through the tissue.However, it is difficult to find a part of the body which is opticallythin enough to pass near infrared light through, especially at thelonger wave lengths. Thus, the preferred method for measurement in thepresent invention is to focus on the reflectance of light from thesample.

Photons reflect and refract at refractive index discontinuities, and solight impinging on tissue immediately has a small reflectance at thetissue surface. This is referred to as specular reflectance. Since thislight does not penetrate into the tissue, it contains little informationabout the tissue constituents. This is especially true in light of thephysiology of skin, which possess an outward layer which is essentiallydead and lacks concentration values of the analytes generally consideredof interest in a sample. Thus, reflected light energy containing analyteinformation is that light which is reflected back to the surface throughrefractive index discontinuities deeper within the tissue sample. Thisreflected light energy is referred to as diffusely reflected light.

Applicants have found that a large fraction of incident photons areabsorbed in the tissue. Those photons which are available for couplingback out of the tissue are likely diverted in their angular path. Infact, by definition, a photon must change direction in order to exit thetissue in a direction towards the input optic. Applicants, however, havefound that a large problem associated with detection is associated withthe refractive index discontinuity between the average tissue refractiveindex and the refractive index of air outside of the tissue. It has beenfound that this discontinuity acting on incident light leads to arefraction and a small specular reflectance of less than about 5percent. However, on the way out, the discontinuity gives rise to acritical angle phenomenon. Because the photon is traveling from a highrefractive index medium to a lower one, a critical angle exists abovewhich a photon is totally internally reflected and will not escape thetissue sample. This critical angle for photons traveling from tissue toair has been found to be about 46 degrees, which presents a problem. Aphoton normally incident on the tissue surface must deviate through alarge angle to exit. Because of the forward directionality ofscattering, this is difficult for a photon to do, and it is very likelyto make a grazing or high angle incidence with the tissue and airinterface. The grazing incidence photons will not escape because thecritical angle is exceeded.

Applicants have found a solution for the differences in refractive indexassociated with coupling light energy exiting tissue to an analyticalinstrument. The solution is the use of an immersion fluid which has verylow absorptivity in the spectral range of interest, and has a viscositycompatible with good flow and coverage, while having a refractive indexwhich closely matches tissue. In preferred embodiments, the indexmatching fluid is preferably minimally or essentially non-absorbing oflight energy in the wavelengths relevant to the blood analyte understudy. The fluid is thus non-spectroscopically active at desiredwavelengths. However, it is believed a minimally absorbingindex-matching fluid, for example one that absorbs less than about 10%of the light energy of analyte relevant wavelengths, could still beutilized. A preferred material is a fluorinated, chlorinated hydrocarbonpolymer oil manufactured by Occidental Chemical under the tradenameFLUOROLUBE. FS5 is a preferred FLUOROLUBE. These oils have a refractiveindex of about 1.38, are non-toxic, and Applicants have found that ithas a spectral signature in the near infrared region which is minimal.

Now referring to FIGS. 1 and 2, partial cross-sectional views of twopreferred embodiments of an apparatus for non-invasively measuring ablood analyte concentration are depicted. The depictions in FIGS. 1 and2 are schematic to depict the concept of utilizing an index-matchingmedium 22 in conjunction with a non-invasive sensor element 11operatively connected to an energy source 16 and a spectrum analyzer 30.The relative size, shape and detail of physical components are notdepicted. The apparatus depicted in FIG. 1 and the apparatus depicted inFIG. 2 generally include three elements, an energy source 16, a sensorelement 11, and a spectrum analyzer 30. The embodiment of FIG. 1 depictsthe sensor element as including an input element 20 and an outputelement 26, which can include a single lens system for both input andoutput light energy. The input element 20 and output element 26 are incontact with a common skin surface 12 of an analyte-containing tissue10. The alternative embodiment of FIG. 2 depicts an alternative sensorelement 11 arrangement, wherein the input element 20 and output element26 are arranged on opposing surfaces 12, 14 of an analyte containingtissue 10. Both embodiments function to give a measure of the absorptionof infrared energy by the analyte-containing tissue 10. However, theembodiment of FIG. 1 is utilized to measure the quantity of light energywhich is reflected from the analyte-containing tissue 10 by the analytecomponents therein. In contrast, the embodiment of FIG. 2 measures thetransmission of light energy through the analyte-containing tissue 10.In either embodiment, the absorption at various wavelengths can bedetermined by comparison to the intensity of the light energy from theenergy source 16.

The energy source 16 is preferably a wide band, infrared black bodysource. The optical wavelengths emitted from the energy source 16 arepreferably between 1.0 and 2.5 μm. The energy source 16 is operativelycoupled to a first means for transmitting infrared energy 18 from theenergy source to the input element 20. In preferred embodiments, thisfirst means 18 is simply the transmission of light energy to the inputelement 20 through air by placing the energy source 16 proximate theinput element 20.

The input element 20 of the sensor element 11 is preferably an opticallens which focuses the light energy to a high energy density spot.However, it is understood that other beam focusing means may be utilizedin conjunction with the optical lens to alter the area of illumination.For example, a multiple lens system, tapered fibers, or otherconventional optical beam-shaping devices could be utilized to alter theinput light energy.

In both embodiments depicted in FIGS. 1 and 2, an output sensor 26 isutilized to receive reflected or transmitted light energy from theanalyte containing tissue 10. As described in conjunction with a methodof analysis below, the embodiment of FIG. 1 has an output sensor 26which receives reflected light energy, while the embodiment of FIG. 2includes an output sensor 26 which receives transmitted light throughthe analyte-containing tissue 10. As with the input element 20, theoutput element 26 is preferably an optical lens. Other opticalcollection means may be incorporated into an output element 26, such asa multiple lens system, tapered fiber, or other beam-collection means toassist in directing the light energy to the spectrum analyzer 30.

A second means for transmitting infrared energy 28 is operativelyconnected to the output element 26. The light transmitted through thesecond means for transmitting infrared energy 28 is transmitted to thespectrum analyzer 30. In a preferred embodiment, the operativeconnection to the output element includes transmission of the reflectedor transmitted light energy exiting the output element through air tothe spectrum analyzer 30. A mirror or series of mirrors may be utilizedto direct this light energy to the spectrum analyzer. In a preferredembodiment, a specular control device is incorporated to separate thespecular reflected light from diffusely reflected light. This device isdisclosed in co-pending and commonly assigned application Ser. No.08/513,094, filed Aug. 9, 1995, and entitled “Improved DiffuseReflectance Monitoring Apparatus”, now U.S. Pat. No. 5,636,633, issuedJun. 10, 1997, the disclosure of which is incorporated herein byreference.

In practicing the method of the present invention, an analyte-containingtissue 10 area is selected as the point of analysis. This area caninclude the skin surface 12 on the finger, earlobe, forearm, or anyother skin surface. Preferably, the area for sampling includes bloodvessels near the surface, and a relatively smooth, uncalloused surface.A preferred sample location is the underside of the forearm.

A quantity of an index-matching medium 22, whether fluid or deformablesolid, is then placed on the skin surface 12 in the area to be analyzed.The sensor element 11, which includes the input element 20 and theoutput element 26, as depicted in the embodiment of FIG. 1, is thenplaced in contact with the index-matching medium 22. Alternatively, aquantity of index-matching medium 22 can be placed on the sensor element11, which is then placed in contact with the skin surface 12 with theindex-matching medium 22 disposed therebetween. In either procedure, theinput element 20 and output element 26 are coupled to theanalyte-containing tissue 10 or skin surface 12 via the index-matchingmedium 22. The coupling of the sensor element 11 with the skin surfacevia the index-matching medium 22 eliminates the need for light energy topropagate through air or pockets of air due to a space between the probeand the skin surface 12 or irregularities in the skin surface 12.

In analyzing for the concentration of glucose in the analyte-containingtissue 10, light energy from the energy source 16 is transmitted throughthe first means for transmitting infrared energy 18 into the inputelement 20. The light energy is transmitted from the input element 20through the index-matching medium 22, to the skin surface 12. The lightenergy contacting the skin surface 12 is differentially absorbed by thevarious components and analytes contained below the skin surface 12 withthe body (i.e., blood within vessels) therein. In a preferredembodiment, the non-absorbed light energy is reflected back to theoutput element 26 upon propagating again through the index-matchingmedium 22. The non-absorbed light energy is transmitted via the secondmeans for transmitting infrared energy 28 to the spectrum analyzer 30.

In the alternative embodiment of FIG. 2, the input element 20 is placedin contact with a first quantity of index-matching medium 22 on a firstskin surface 12, while the output element 26 is placed in contact with asecond quantity of index-matching medium 24 on an opposing skin surface14. As with the previous embodiment, the index-matching medium 22 can befirst placed on the input element 20 and output element 26 prior tocontact with the skin surface 12. With this alternative embodiment, thelight energy propagated through the input element 20 and first quantityof index-matching medium 22 is differentially absorbed by theanalyte-containing tissue 10, while a quantity of the light energy atvarious wavelengths is transmitted through the analyte-containing tissue10 to the opposing or second skin surface 14. From the second skinsurface 14, the non-absorbed light energy is propagated through thesecond quantity of index-matching medium 24 to the output element 26with subsequent propagation to the spectrum analyzer 30 for calculationof the analyte concentration.

As previously stated, the index-matching medium 22 of the presentinvention is a key to the improved accuracy and repeatability of themethod described above. The index-matching medium can preferably be afluid composition containing chlorofluorocarbons. The composition canalso be a mixture of chlorofluorocarbons and perfluorocarbons. Apreferred composition includes chlorotrifluoroethylene. A preferredcomposition contains about 80% to about 99.8% by weight ofchlorofluorocarbons. As previously stated, the present inventionutilizes an index-matching fluid to optimize the input and output oflight energy to and from a sample containing an analyte of interest tobe measured. In its broadest sense, the index-matching fluid of thepresent invention can be any fluid which creates an improved opticalinterface over that interface which results from simply placing theprobe of the present invention on a skin surface. Absent theindex-matching fluid of the present invention, this interface caninclude gaps which are air filled and cause detrimental refraction oflight both going into the tissue and exiting the tissue. Thus, anyindex-matching fluid having a refractive index closer to that of thetissue at about 1.38 versus the refractive index of air of about 1.0would provide an improved interface.

Applicants have also recognized that the usefulness of the apparatus ofthe present invention requires that the coupling of the sensor berepeatable and that the results be an accurate reflection of the bloodglucose level of the patient. To this end, Applicants have found that itis preferable for the index-matching fluids of the present invention tocontain diagnostic additives and/or physiological additives. Thediagnostic additives provide an assessment of the quality of the lens totissue interface and/or an assessment of the instrument's presentperformance, while the physiological additives alter the physiology ofthe tissue to correct for differences in tissue analyte concentrationversus blood analyte concentration. A discussion of these additivesfollows.

The non-invasive measurement of glucose in tissue by the presentinvention is improved by placing an additive into the index-matchingfluid that allows evaluation of the thickness of the fluid when thetissue is placed in contact with the instrument. In preferredembodiments, the additive also provides a calibration of the instrumentby including a compound of known high absorption at a specifiedwavelength of light. Such additives also further assure that the correctindex-matching fluid is being utilized for the instrument.

Since an index-matching fluid inherently causes a change of height inthe tissue above the sample probe, the measurement of this height canaid in the overall glucose or other analyte measurement, while allowinga path length correction to be applied to the spectral measurement as afunction of the tissue height above the sampler. This can insure areproducible, consistent height is achieved before commencing thespectral measurement of the tissue, and further allows for theadjustment of the height before commencing the spectral measurement ofthe tissue. In this way, the user can be certain that spurious resultsare not achieved due to excess matching fluid height, insufficientindex-matching fluid being utilized, or some other misplacement of thetissue surface relative to the analyzer.

Laboratory spectrometers utilize a Fourier Transform system whichincorporates a laser reference signal to establish the wavelengths andguarantees that the instrument is calibrated. However, it is likelyinstruments that are affordable for an end user will not use a laser,but rather will be dispersion type instruments such as gratings, CCDarrays and others. With such instruments, it is important to makecertain that calibration is proper prior to each analysis of bloodanalyte. To this end, Applicants have found that the addition of anadditive which includes a well-defined spectral feature at a knownwavelength of light can be utilized to assure calibration.

The use of a known spectrally active additive to the index-matchingfluid also insures that the end user is using a correct index-matchingfluid for which the instrument has been calibrated and programmed. Theuse of a different index-matching fluid could result in an error in thenon-invasive analyte measurement by absorbing light energy in the areasof interest for the particular analyte.

To accomplish the above repeatability, accuracy and quality assurance, aspectroscopically active agent is preferably added to the index-matchingfluid. The agent preferably has sharp bands of absorption outside theregion of interest to measure the blood analyte. For example, in apreferred method for glucose analysis, the agent would be active outsidethe ranges of 4200-4900 and 5400-7200 wave numbers. The agent could alsobe active within this range so long as there is no significant overlapwith wavelengths actually used to calculate glucose concentration. Theadditive can be manufactured by placing an appropriate functional groupon perfluorinated hydrocarbons. The perfluorinated hydrocarbons arespectrally inactive in the region of interest, however, the functionalgroup placed upon the perfluorinated hydrocarbons may be spectrallyactive. Further, these functional groups do not interfere with theanalysis of the blood analyte of interest. Exemplary compounds includeperfluoro-2-butyltetrahydrofuran and perfluorosuccinyl chloride.

In an alternative embodiment, the index-matching fluid and diagnosticadditive can comprise the same fluid which provides both functions. Forexample, perfluoro-2-butyltetrahydrofuran can be utilized as anindex-matching medium which improves the optical interface, and at thesame time includes a functional group which makes the compoundspectrographically active in a desired range for diagnostic purposes.

The near infrared light energy of the present invention is preferablyutilized to measure a blood analyte such as glucose. However, the lightenergy interrogates the skin as a whole, while the blood vessels make upless than 10% of the skin volume. Therefore, in reality the total skinglucose content is being used as a surrogate for blood glucoseconcentration. This fact can lead to inaccurate test results if there isa large difference between the tissue glucose concentration and theblood vessel glucose concentration, such as in times of rapidly risingor falling blood glucose levels. Blood glucose can rise acutely after ameal or during glucose production by the liver, while there is acommensurate but lagged rise of the skin glucose concentration. Thislag, due to the finite time required for the glucose to diffuse into thegreater skin water compartment, can take minutes to tens of minutesdepending upon the magnitude of the rise and the surface area of thecapillaries available for diffusion. Applicants have found that byincreasing the superficial skin capillary blood flow in the area ofanalysis, the surface area of the capillaries increases and the rate ofdiffusion of glucose from the vessels into the skin also increasessignificantly. This markedly results in reduced equilibration times anda significant reduction in measurement error attributable to thedisequilibrium between blood glucose and skin water glucoseconcentration during periods of changing blood glucose concentration.

Applicants have found that vasodilating agents which are topicallyapplied can provide the improved equilibration. These agents work bydiffusing into the skin and blocking the adrenergic receptors on thesmall arterioles that feed the capillary vessels. This results indilation of the arterial sphincters, a reduction of resistance to flow,and an increase in pressure and size of the capillaries. A number ofpreferred vasodilating agents include: methyl nicotinamide, minoxidil,nitroglycerin, histamine, menthol, and capsaicin.

The compound can contain a hydrophilic additive, such as isopropylalcohol. The hydrophilic additive is believed to tie up the moisture inthe skin surface to improve the interface between the medium and theskin. Further, the index-matching medium can contain cleansing agents tobind the oil in the skin at the sample point and reduce the effectthereof. A surfactant can also be included in the composition. Thesurfactant improves the wetting of the tissue, thus improving contact.Finally, an antiseptic compound can be added to the index-matchingmedium.

In an alternative embodiment of the current invention, the indexmatching between the optical sensor elements and the tissue can beperformed by a deformable solid. The deformable solid can alter itsshape such that air gaps, due in part to the uneven surfaces of theskin, are minimized. Deformable solids can include at least gelatin,adhesive tape, and substances that are liquid upon application butbecome solid over time.

The index-matching medium, preferably has a refractive index of1.30-1.45, more preferably from 1.35-1.40. Utilization of a refractiveindex in this range has been found to improve the repeatability andaccuracy of the above method. It is recognized that the refractive indexof the index-matching medium must be consistent throughout thecomposition to prevent refraction of light energy as it passes throughthe medium. For example, there should be no air bubbles present in theindex-matching medium which could cause a discontinuity in refractiveindex.

In a preferred embodiment, the concentration of glucose in the tissue isdetermined by first measuring the light intensity received by the outputsensor. These measured intensities in combination with a calibrationmodel are utilized by a multivariate algorithm to predict the glucoseconcentration in the tissue. The calibration model empirically relatesthe known glucose concentrations in the calibration samples to themeasured intensity variations obtained from said calibration samples. Ina preferred embodiment, the multivariate algorithm used is the partialleast squares method, although other multivariate techniques can beemployed.

The input infrared energy from the input element sensor is coupled tothe analyte-containing sample or blood through the index-matching medium22. There is, thus, differing absorption at several wavelengths of theinfrared energy as a function of the composition of the sample. Thediffering absorption causes intensity variations of the infrared energypassing through the analyte containing samples. The derived intensityvariations of the infrared energy are received by reflectance ortransmittance through the analyte-containing sample by the outputelement of the sensor, which is also coupled to the blood oranalyte-containing sample through the index-matching medium 22.

The spectrum analyzer 30 of the present invention preferably includes afrequency dispersion device and photodiode array detectors inconjunction with a computer to compare the data received from suchdevices to the model discussed above. Although preferable, other methodsof analyzing the output energy may be utilized.

The frequency dispersion device and photodiode array detectors arearranged so that the array includes multiple output leads, one of whichis assigned to a particular wavelength or narrow range of wavelengths ofthe energy source 16. The amplitude of the voltage developed on each ofthe leads is commensurate with the intensity of the infrared energyincident on each particular detector in the array for the wavelength ofthe source associated with that detector. Typically, the photodiodes ofthe array detector are passive, rather than photovoltaic, althoughphotovoltaic devices may be employed. The diodes of the array detectormust be supplied with DC power supply voltage as derived from a powersupply and coupled to the diodes of the array detector via a cable. Theimpedance of the diode elements of the array detector are changed as afunction of the intensity of the optical energy incident thereon in thepass band of the energy source 16 associated with each particularphotodiode element. The impedance changes can control the amplitude ofthe signal supplied by the array detector to a random access memorycomputer.

The computer includes a memory having stored therein a multivariatecalibration model empirically relating the known glucose concentrationin a set of calibration samples to the measure intensity variations fromsaid calibration samples, at several wavelengths. Such a model isconstructed using techniques known by statisticians.

The computer predicts the analyte concentration of theanalyte-containing sample 10 by utilizing the measured intensityvariations, calibration model and a multivariate algorithm. Preferably,the computation is made by the partial least squares technique asdisclosed by Robinson et al. in U.S. Pat. No. 4,975,581, incorporatedherein by reference.

It is has been found that considerable improvement in detectionprecision is obtained by simultaneously utilizing at least severalwavelengths from the entire spectral frequency range of the energysource 16 to derive data for a multivariate analysis. The multivariatemethod allows both detection and compensation for interferences, thedetection of meaningless results, as well as for modeling many types ofnon-linearities. Since the calibration samples used to derive the modelshave been analyzed on a multivariate basis, the presence of unknownbiological materials in the analyte containing tissue 10 does notprevent or distort the analysis. This is because these unknownbiological materials are present in the calibration samples used to formthe model.

The partial least squares algorithm, calibration model and measuredintensity variations are employed by the computer to determine theconcentration of the analyte in the analyte containing tissue 10. Theindication derived by the computer is coupled to conventionalalphanumeric visual displays.

Experimental

Comparative testing was conducted to document the effect of utilizing anindex-matching medium versus no index-matching medium on the sameapparatus. Reference should be made to FIG. 3 which is a graphicalrepresentation of the results of the experiment, wherein line 50represents analysis without the index-matching medium, and line 52documents the improved accuracy of the result when the sensor element iscoupled to the skin surface via an index-matching medium. To conduct thetest, forearm sampling was conducted with and without the index-matchingmedium with a two minute time resolved data collection.

The apparatus utilized to conduct the experiment included a Perkin-Elmer(Norwalk, CT) System 2000 Fourier Transform Infrared Spectrometer (FTIR)with a 4 mm DIA indium antimonide (InSb) single element detector. Thelight source was a 100 watt quartz tungsten halogen light bulb fromGilway Technical Lamp (Woburn, Mass.). The interferometer employed aninfrared transmitting quartz beamsplitter. Data collection was via atransputer link to a PC running Perkin-Elmer TR-IR software. Datavisualization was accomplished in Matlab (MathWorks, Natick, Mass.).Sampling optics were constructed in-house and consisted, in part, of theoptical system described in co-pending application 08/513,094, filedAug. 9, 1995, entitled “Improved Diffuse Reflectance MonitoringApparatus”, now U.S. Pat. No. 5,636,633, issued Jun. 10, 1997. Allinstrument parameters were identical for the collection of both spectra.

The experimental procedure was as follows. The sampling surfaceconsisted of a MgF₂ hemisphere mounted with its radiused side facingdownward, and its flat surface placed horizontally. Light was launchedinto the hemisphere from below. The flat surface of the hemisphere, themount for the hemisphere, and the holder for the mount all comprised aflush, horizontal sampling surface. The patient's arm was placed down onthis surface, such that the underside of the forearm rested against thehemisphere sampling surface. The forearm area had previously been shavedand washed with soap and water, then swabbed with isopropyl alcohol. Thearm was then covered with a blood pressure cuff which was inflated to apressure of 30 mm Hg. The cuff acted to hold the arm in place and toprevent motion of the arm relative to the hemisphere. The samplingsurface was held at a constant temperature of 28 C. by resistance heaterelements and a thermocouple feedback device. After the arm was situatedin the device, it was allowed to equilibrate for 30 seconds prior tosampling.

Referring to FIG. 3, the top trace, labeled 50, shows the resultobtained when sampling in the previously described mode in the absenceof index-matching medium. In the bottom trace, labeled 52, 100microliters of chlorotrifluoroethene was applied to the surface of thehemisphere prior to placing the arm. There are several notabledifferences. Most apparent is the spread of the data. 50 and 52 are eachcomprised of multiple spectra. With FLUOROLUBE, all of the spectraoverlay each other quite closely. This indicates that the interface isquite stable. Without FLUOROLUBE, the interface is extremely unstable.Also, notable is the data near 5200 cm⁻¹. This is the position of thestrongest water band. Without FLUOROLUBE, this band appears weaker,since it is contaminated with specular light. In fact, note that thespread of the data is largest under this band. In fact, the differencebetween the two traces can be attributed largely to spurious energy fromspecular contamination.

New characteristics and advantages of the invention covered by thisdocument have been set forth in the foregoing description. It will beunderstood, however, that this disclosure is, in many respects, onlyillustrative. Changes may be made in details, particularly in matters ofshape, size, and arrangement of parts, without exceeding the scope ofthe invention. The scope of the invention is, of course, defined in thelanguage in which the appended claims are expressed.

What is claimed is:
 1. A method for evaluating the separation of asensor element for spectrographic analysis relative to tissue underanalysis comprising the steps of: providing an apparatus forspectrographic analysis, said apparatus including an energy sourceemitting energy at multiple wavelengths, including selected wavelengthsrelevant to measuring an attribute of interest in said tissue, an inputelement, an output element, and a spectrum analyzer; providing a medium,said medium having a known spectral absorbance, and disposing a quantityof said medium between said tissue and said input element and outputelement to couple said elements to said tissue through said medium;irradiating said tissue through said input element with said multiplewavelengths of energy; and collecting at least a portion of said energywith said output element and evaluating the separation of the sensorelement based on the spectral absorbance of the medium.
 2. The method ofclaim 1, wherein evaluating the separation of the sensor element basedon the spectral absorbance of the medium comprises evaluating thedistance between the tissue and the sensor element.
 3. The method ofclaim 1, further comprising the step of deriving a pathlength correctionfor the analysis based on the evaluated separation of the sensorelement.
 4. The method of claim 1, wherein said medium is a fluidcomposition comprising about 0.2% to about 20% of a compound having aknown spectral absorbance.
 5. The method of claim 1, further comprisingthe step of determining the attribute of the interest only when propercoupling is confirmed.
 6. A method for evaluating instrument calibrationduring non-invasive determination of an attribute of tissue comprisingthe steps of: providing an apparatus for spectrographic analysis oftissue, said apparatus including an energy source emitting energy atmultiple wavelengths, including selected wavelengths relevant to saidattribute of tissue, an input element, said apparatus further includingan output element and a spectrum analyzer; providing a medium, saidmedium having known spectral absorbance, and disposing a quantity ofsaid medium between said tissue and said input element and outputelement to couple said elements to said tissue through said medium;irradiating said tissue through said input element; and collecting atleast a portion of the energy with said output element and evaluatinginstrument calibration utilizing the spectral absorbance of said medium.7. The method of claim 6, further comprising the step of determiningsaid attribute of tissue only after instrument calibration is confirmed.8. The method of claim 6, wherein said medium is a fluid compositioncomprising about 0.2% to about 20% of a compound having said spectralabsorbance.
 9. The method of claim 6, wherein substantially all of saidmedium comprises a compound having said known spectral absorbance.